Following vesicle fusion and exocytosis of neurotransmitter, vesicular retrieval is critical to allow transmission to continue during extended activity. The demands on the vesicle cycle are particularly great in tiny nerve terminals that have a limited number of vesicles. Classical full-collapse fusion (FCF) proceeds by vesicles completely flattening into the plasma membrane, thereby releasing their transmitter, but also completely losing their lipid and protein content and spherical shape. Vesicles are then reconstructed via clathrin- mediated membrane retrieval. Another set of phenomena generically known as "kiss and run" (K&R) has been proposed wherein vesicular identity is maintained and the same vesicle is repeatedly reused. The likely consequence is increased synaptic efficiency and information throughput. To gain further insights into these apparently distinct fusion modes, the following specific aims are proposed: (1) To describe fundamental properties of modes of fusion/retrieval using novel optical approaches. New measurements will indicate the time course by which vesicles undergo fusion for the first time after a period of rest and provide a simple way of dissecting the contributions of FCF and K&R. Quantum dots (QDs) will be used to specifically register FCF events. Modification of QDs by conjugation with the pH-sensitive dye Flubi2 will permit real-time, single vesicle tracking of both K&R and FCF. (2) To clarify key structural determinants of fusion/retrieval modes. The new techniques will allow examination of the cell biological and molecular factors that govern the balance between K&R and FCF. Do vesicles in the readily releasable pool (RRP) continue to stay in the RRP over multiple bouts of release? Can the concept of vesicle reuse be demonstrated definitively? The location of RRP and total recycling pool (TRP) vesicles will be examined. Candidate molecular mechanisms for regulation of fusion/retrieval modes involving dynamin-1 and -2 will be tested. (3) To determine the impact of fusion/retrieval modes on quanta! neurotransmission. Single quantal events during K&R and FCF will be separately analyzed by recording excitatory postsynaptic currents in combination with imaging of fusion/retrieval events. This work will clarify how vesicles are deployed for efficient transmission in nerve terminals and how this usage is modified by parameters such as resting potential and firing pattern. Such questions are central to understanding synaptic communication in both healthy and diseased neural circuits.